WO2014015804A1 - 利用电荷排斥作用制备同二聚体蛋白混合物的方法 - Google Patents
利用电荷排斥作用制备同二聚体蛋白混合物的方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
- C07K2317/526—CH3 domain
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to a process for the preparation of a mixture of homodimeric proteins, and in particular to a process for the preparation of a mixture of homodimeric proteins by charge repulsion.
- the invention also relates to the use of a homodimeric protein mixture prepared by the method, and the use of the method to prepare a mixture of homodimeric proteins. Background technique
- Monoclonal antibody drugs have grown rapidly in the last fifteen years and have become the growth point of the pharmaceutical industry. Since 1996, a total of 30 or so monoclonal antibodies have been approved for marketing. Among them, nine monoclonal antibodies have annual sales of more than one billion US dollars. The total sales of monoclonal antibodies in 2010 exceeded $30 billion, and the annual growth rate exceeded 10%. Because of the high target specificity of monoclonal antibodies, only a single target can be inhibited. In many diseases such as tumors and autoimmunity, it is necessary to suppress multiple signaling pathways to avoid compensatory effects. For viral infections, due to the high mutation rate of the virus, it is often necessary to inhibit multiple antigenic sites to prevent escape. Therefore, there are several alternatives that can solve such problems.
- An alternative is to use a polyclonal antibody, or to obtain a heterodimer such as a silent specific antibody by engineering the Fc portion of the antibody.
- Yet another approach is to treat with a mixture of antibodies, which may comprise two or more antibodies directed against different epitopes on the same target, or a mixture of antibodies directed against different targets.
- US7262028 describes a method for producing a bivalent antibody or a mixture of bivalent antibodies by cloning a single host cell by expressing a light chain and different heavy chains, and provides a method for generating an antibody combination, which can be combined Screening is useful in a variety of applications.
- WO/2010/084197 describes a method for producing a mixture comprising two or more different antibodies from a single host cell clone. In one embodiment, it is a mixture of different monovalent antibodies. In another embodiment, it is a mixture of monovalent and bivalent antibodies, and the method is controlled by the exchange between the arms of the natural Fab of IgG4, and finally changes the hinge region of the phenomenon and the partial acid of CH3, and stabilizes the same Dimer, but the patent does not completely solve the problem of heterodimerization.
- a method for expressing a polyclonal antibody library has been described in US Pat. No. 5,789,208 and U.S. Patent No. 6,335,163, the disclosure of which is incorporated herein by reference.
- the selected heavy and light chain variable region gene combinations are transferred in bulk to a eukaryotic expression vector providing a constant region gene in a strand pairing manner to obtain a sub-library of intact polyclonal antibodies.
- a eukaryotic expression vector providing a constant region gene in a strand pairing manner to obtain a sub-library of intact polyclonal antibodies.
- stable cloning produces polyclonal antibodies that can be mixed to obtain a mixture of monoclonal antibodies.
- it is theoretically possible to obtain polyclonal antibodies directly from a recombinant production process by using this method by culturing a mixed population of transfected cells the stability of the mixed cell population and the resulting polyclonal antibodies are thus There may be potential problems with consistency.
- Sym004 is a novel in vitro antibody mixture that targets the epidermal growth factor receptor (EGFR).
- Sym004 consists of two antibodies that block ligand binding, receptor activation, and downstream signaling, and is thought to induce EGFR clearance from cancer cells by inducing EGFR invagination and degradation.
- mCRC metastatic colorectal cancer
- SCCHN head and neck squamous cell carcinoma
- Symphogen A/S's antibody mixture technology firstly obtains multiple antibodies against the same target by screening the antibody screening platform, then constructs the molecular structure of each antibody, separately shakes the cells, mixes them, and finally expands the mixture gradually. And carry out purification process optimization to obtain the final product.
- this method uses multiple recombinant host cells to produce a mixture of multiple homomeric proteins, and there is still an unstable cell growth rate and antibody production rate. The problem. Since this method expresses a separate antibody by a single cell, there is no problem of heterodimer.
- the inventors of the present invention have surprisingly discovered a method of simultaneously preparing two or more proteins or antibodies in a recombinant cell by a large number of experiments, thereby completing the present invention, and specifically include the following aspects:
- a first aspect of the invention relates to a method for obtaining a mixture comprising two or more proteins using a recombinant host cell, the protein being in the form of a dimer of a monomeric chain and a monomeric chain, and the two Or two or more proteins comprising the same domain, characterized in that the method comprises replacing a partial amino acid of two monomeric chains in the same domain of one or more proteins with oppositely charged
- the step of amino acid makes it difficult to form heterodimers due to the rejection of the same charge between the monomer chains of different proteins, and the monomer chains of the same protein are easier due to the attraction of opposite charges. Form a homodimer.
- the protein is a fusion protein consisting of an antibody or a portion of an antibody.
- a method according to any one of the first aspects of the invention wherein the at least one protein of the amino acid is not replaced.
- the amino acid of one of the proteins is not replaced and the amino acid of the other protein is replaced.
- the acid of both proteins is replaced.
- the acid exchanged between the different proteins has at least one different position, preferably, the acid substituted between the different proteins.
- the location is different.
- a method according to any one of the first aspects of the invention, wherein said same domain means The Fc region of the antibody.
- said antibody is selected from I g G (e.g. IgGl, IgG2, IgG3), IgA (e.g. IgAl, IgA2), IgE, IgD , IgM (e.g. IgMl, IgM2) .
- Ig G e.g. IgGl, IgG2, IgG3
- IgA e.g. IgAl, IgA2
- IgE IgE
- IgD IgD
- IgM e.g. IgMl, IgM2
- replacing the partial amino acid in the same domain with the oppositely charged amino acid comprises the steps of:
- one or more pairs for example, two pairs, three pairs, four pairs of
- the charged acid in the positive and negative charge paired charged amino acids obtained in the step (2) replacing the selected charged amino acid It is an oppositely charged charged amino acid.
- the charged amino acid is selected from the group consisting of lysine (lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu).
- the same domain refers to the Fc region or CH3 domain of the antibody, and the positively and negatively charged paired charged amino acids are selected from the pairings shown in a)-h) below.
- Lys at position 392 of one of the proteins was replaced by Asp, Lys at position 409 was replaced with Asp, and Asp at position 399 was replaced with Lys.
- the positively and negatively charged paired charged amino acid is in the sequence shown in SEQ ID NO: 2 at the position of 8 pairs of tyrosine acids as shown in a) -hi):
- the position in the sequence shown by SEQ ID NO: 4 is 8 pairs of amino acids as shown by a2)-h2): a2) Glu (E) at position 399 of the first chain and Lys (K1) at position 482 of the second chain B2) the 400th Glu (E) of the first strand and the 413th Lys (K) of the second strand; c2) the 413th Lys (K) of the first strand and the 400th Glu of the second strand (E) ; d2 ) Lys ( K) at position 435 of the first strand and Asp ( D ) at position 442 of the second strand; el) 442th Asp (D) of the first strand and 435 of the second strand Bit Lys (K); f2) The 442th Asp (D) of the first strand and the 452th Lys (K) of the second strand; g2) The 452th Lys (K) of the first strand and the second strand The 442th Asp(D);
- the method of replacing a partial amino acid in the same domain with an oppositely charged amino acid comprises: obtaining a nucleotide encoding a protein of the amino acid replacement protein Sequence, the coding nucleotide sequence is expressed by a recombinant host cell to obtain a protein after acid substitution.
- different proteins can be separately cloned into an expression vector, and different expression vectors can be co-transfected into a host cell, a recombinant host cell can be cultured, and a protein can be expressed to obtain a protein mixture; and different proteins can also be operably
- the ligation is cloned into the same expression vector, transferred to a host cell, and cultured to obtain a protein mixture.
- a second aspect of the invention relates to a mixture comprising two or more proteins which are present in the form of a dimer of a monomeric chain and a monomeric chain, and wherein the two or more proteins contain the same a domain characterized in that one of the two monomeric chains of one or more proteins in the same domain is replaced by an oppositely charged amino group.
- the acid makes it difficult to form heterodimers due to the repulsive action of the same charge between the monomer chains of different proteins, and the monomeric chains of the same protein are more likely to form homodimerization due to the attraction of opposite charges. body.
- the protein is a fusion protein consisting of an antibody or a portion of an antibody.
- the amino acid of one of the proteins is not replaced and the acid of the other protein is replaced.
- the acid of both proteins is replaced.
- the acid exchanged between the different proteins has at least one different position, preferably, the acid substituted between the different proteins.
- the location is different.
- the antibody is selected from the group consisting of IgG (e.g., IgG1, IgG2, IgG3), IgA (e.g., IgAl, IgA2), IgE, IgD, IgM (e.g., IgMl, IgM2).
- IgG e.g., IgG1, IgG2, IgG3
- IgA e.g., IgAl, IgA2
- IgE e.g., IgAl, IgA2
- IgE IgE
- IgD IgD
- IgM e.g., IgMl, IgM2
- the partial amino acid is an interface between the same domains of two monomer chains of the protein, preferably, the interface amino acid is positive Negatively charged paired charged amino acids; more preferably, one or more pairs (e.g., two pairs, three pairs, four pairs) of paired amino acids are replaced with oppositely charged charged amino acids.
- the charged amino acid is selected from the group consisting of lysine (lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu).
- the same domain refers to the Fc region of an antibody or The CH3 domain
- the positively and negatively charged paired charged acyl acid at this time is selected from the group consisting of the following aryl acids shown in a)-h):
- Lys at position 392 of one of the proteins was replaced by Asp, Lys at position 409 was replaced with Asp, and Asp at position 399 was replaced with Lys.
- the positively and negatively charged paired charged amino acid is in the sequence shown in SEQ ID NO: 2 at the position of 8 pairs of acid as shown in a) -hi):
- the charged amino acid of the positive and negative charge pairing position in the sequence shown in SEQ ID NO: 4 is 8 pairs of amino acids as shown by a2)-h2): a2) the first strand Glu (E) at position 399 and Lys (K) at position 482 of the second chain; bl) Glu (E) at position 400 of the first chain and Lys (K) at position 413 of the second chain; c2) Lys (K) at position 413 of the first strand and Glu (E) at position 400 of the second strand; dl) Lys (K) at position 435 of the first strand and Asp (D) at position 442 of the second strand; el The 442th Asp (D) of the first strand and the 435th Lys (K) of the second strand; f2) the 442th Asp (D) of the first strand and the 452th Lys (K) of the second strand ; g2 ) Lys
- a third aspect of the invention relates to a mixture of proteins obtained according to the method of any of the first aspects of the invention.
- a polypeptide comprising a CH3 region forms an interaction interface due to an interaction force between amino acids and forms a dimer, and the amino acid on the interaction interface of the CH3 region is modified by a charge repulsion effect.
- the charge-based acid is a charge-based acid that forms a charge-repelling effect.
- a positively charged amino acid on the contact surface Acid, arginine, histidine is a negatively charged acid (aspartic acid, glutamic acid) or vice versa.
- the interaction between the charged amino acids in the pair is obtained, and any one or more of the amino acids are selected to analyze the selected acid.
- the selected amino acids were mutated to charged amino acids, and the effects on the homodimers and heterodimers after mutation were investigated. The mutations were compared with those before the mutations. If the mutation is appropriate, it will produce an effect of enhancing homodimers and weakening the formation of heterodimers. Finally, choose a reasonable amino acid mutation to maximize the enhancement of homodimers and weaken the role of heterodimers.
- the method described above is defined in the specific method as the amino acid of the mutated CH3 region paired with the charged amino acid on the same chain is an oppositely charged amino acid, and the two-chain Fc is still the opposite charge in the process of forming the homodimer.
- the charge attraction caused by the acid maintains the interaction ability, while the Fc is in the process of forming the heterodimer, because the charge of the paired amino acid in one of the chains is electrically exchanged, resulting in the same charge repulsion, and cannot be formed.
- Heterodimer finally obtaining a mixture of Fc antibodies or proteins with only homodimers.
- the protein is also referred to as a polypeptide, and contains 10 or more amino acids, preferably 50 or more amino acids, and more preferably 100 or more amino acids.
- the protein is an antibody or comprises a portion of an antibody.
- the protein is an Fc fragment of IgG1.
- the protein is a fusion protein of a single chain antibody (SrcFv) with an Fc fragment of IgG1.
- the host cell is a cell suitable for expressing the protein or antibody, for example Such as prokaryotic cells or eukaryotic cells.
- the prokaryotic cell is, for example, Escherichia coli; the eukaryotic cell is, for example, a yeast cell or a mammalian cell, such as a human epithelial cell line (such as 293H), a Chinese hamster ovary cell line (CHO) or a myeloma cell. .
- the different species of proteins contain the same domain.
- the same domain refers to the CH3 region of an antibody or the Fc region of an antibody.
- the monomeric chain is also referred to as a single polypeptide, and refers to one of the monomers or a subunit which constitutes a dimeric protein.
- the two monomeric strands constituting the dimer are symmetrical, i.e. the sequences of the two monomeric strands are identical.
- the replacement of the amino acid means replacement of the amino acid at the corresponding position of the two monomer strands constituting the dimeric protein.
- the dimer refers to a combination of two subunits or monomers during formation of a protein or nucleic acid, which may be covalently bonded or non-covalently.
- the domain refers to a region of a biological macromolecule, particularly a protein, having a specific structure and an independent function.
- the domain refers to the CH3 region of an antibody or the Fc region of an antibody.
- the interface amino acid refers to an amino acid in which a domain and a domain are in contact with each other to form a contact interface.
- the acid at the interface may be composed of two basic acids or may be composed of a plurality of amino acids.
- a protein or “isoprotein” refers to a protein sequence which is a protein formed by homologous dimer formation.
- the antibody mixture obtained by the method of the invention may be a protein or antibody mixture of two homodimers, or a protein or antibody mixture of two or more homodimers, preferably two homodimeric proteins or Antibody mixture.
- the domain comprising CH3 may be a separate CH3 region or a human immunoglobulin Fc region comprising a CH3 region.
- the CH3 region of the human immunoglobulin Fc region is derived from wild-type human immunoglobulin. Fc region. Wild-type human immunoglobulin Fc refers to the sequence in which it occurs, and of course the Fc sequence will have some subtle differences in the individual.
- the human immunoglobulin Fc of the present invention also includes a fragment which has an individual change to the amino acid of the wild type human immunoglobulin Fc sequence, for example, a change in some of the local acid in the Fc region, such as including some in glycosylation. Site-mutated amino acids, or other nonsense mutations.
- the sequence of the CH3 domain may, for example, be the sequence of amino acids 148-252 of SEQ ID NO: 2.
- the sequence of the Fc region may be, for example, the sequence shown by amino acids 26-252 of SEQ ID NO: 2.
- the term "human immunoglobulin Fc" refers to the human immunoglobulin chain constant region, particularly the end of or a part of the immunoglobulin heavy chain constant region.
- the immunoglobulin Fc region can comprise a combination of two or more domains of the heavy chain CH1, CH2, CH3, CH4 and an immunoglobulin hinge region.
- the Fc region of IgG corresponds to the "lower hinge" -CH2-CH3 domain (for IgG, CH2 and CH3 are also referred to as Cy2 and Cy3 domains).
- immunoglobulins can be divided into different types, mainly five types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, some of which can be further divided into subclasses (the same species) Type), such as IgG-l, IgG-2, IgG-3, IgG-4, IgA-1 and IgA-2.
- Similar domains of other IgG subclasses can be determined by aligning the heavy or heavy chain fragments of the IgG subclass with the amino acid sequence of the heavy or heavy chain fragment of human IgGl. Selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is well within the purview of those skilled in the art. Because the acid interaction interface between human and mouse is very conservative, charge repulsion for the preparation of homodimeric proteins or antibody mixtures is equally applicable to human or murine IgA, IgD, IgE, IgG and IgM. The same method is also applicable to the mutating of non-charged amino acids to charged amino acids in the CH3 region.
- the numbering of the residues in the Fc region is Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md. (1991), which is hereby incorporated by reference.
- the immunoglobulin heavy chain number in the EU index "The EU index in Kabat, which refers to the number of human IgGl EU antibodies. The position of the acid in the column of the Fc region of the antibody, They are all expressed according to the EU index of Kabat et al.
- the antibody prototype for generating a mixture of homodimeric proteins may be an antibody, an immunoglobulin, an Fc fusion polypeptide, an Fc conjugate (see Fig. 2), and the list is not limitative.
- the homodimeric protein may be a polypeptide homodimer protein comprising an Fc region.
- these include, but are not limited to, antibodies, Fc fusion proteins, Fc conjugates, Fc-derived polypeptides, isolated Fc, and fragments thereof.
- the homodimeric protein can be a naturally occurring polypeptide, a variant of a naturally occurring polypeptide, a modified form of a naturally occurring polypeptide, a synthetic polypeptide or a polypeptide comprising a non-protein fragment.
- a modified form of a naturally occurring polypeptide is a polypeptide that is not encoded by a naturally occurring gene.
- the polypeptide of ⁇ U can be a chimeric antibody or a humanized antibody.
- the homodimeric protein mixture can be purified from the host cells by standard experimental means.
- the homodimeric protein contains an Fc region, it can be purified with protein A.
- Purification methods include, but are not limited to, color transfer techniques such as size exclusion, ion exchange, affinity chromatography, and filtration.
- the method for separating and purifying the homodimer mixture of the present invention also includes a suitable combination of the above various methods.
- the invention also relates to engineered monomeric chains or individual polypeptides that constitute homodimeric proteins or antibodies.
- the invention still further relates to nucleic acid sequences encoding engineered constituent homodimeric proteins or antibodies (or monomeric strands or polypeptides alone).
- the invention further relates to a pharmaceutical composition
- a pharmaceutical composition comprising an engineered homodimeric protein or antibody (or monomeric strand or polypeptide alone).
- the invention provides for the preparation of two or a recombinant host cell A method of mixing two or more proteins or antibodies which increase the homodimer content of the protein or antibody while reducing the amount of other undesirable products such as heterodimers.
- the experimental results show that the protein or antibody mixture component obtained by the invention is pure and reliable.
- the protein or antibody mixture prepared by the invention can simultaneously act on different epitopes of the same target, and can simultaneously inhibit the action of multiple antigens by combining different antigens, and provides a new method and a pathway for the treatment of diseases such as tumors.
- Figure 1 is a schematic view showing the structure of the recombinant vector pcMVp-SP-Fc.
- Figure 2 is a schematic view showing the structure of the recombinant vector pcDNA3.1-zeo-ScFv-Fc.
- FIG. 3 Sequence alignment of human (a) and murine (b) IgG subtypes.
- the CH3 of the heavy chain is compared.
- the star (*) ⁇ acid is an amino acid in the CH3-CH3 interaction region represented by the IgGl human Fc crystal structure, and the square box indicates the preferred acid dimer of the homodimer mixture. It is worth noting that the charge is highly conserved within most IgGs.
- the asterisk (*) in (1)) and (c) is an amino acid in the CH3-CH3 interaction region expressed by human IgG1.
- Figure 4 shows a schematic example of the wild-type charge interaction effect and the charge interaction in the mutant to prevent heterodimers and support homodimer formation.
- charge interactions simultaneously support the formation of heterodimers and homodimers.
- One of the strands Fc CH3 region D399K and K409D double mutations are unable to form heterodimers due to charge repulsion effects, and at the same time, homodimer mixtures are easily formed due to charge attraction.
- Figure 5 shows the results of electrophoresis detection of homodimers (ScFv-Fc/ScFv-Fc, Fc/Fc) and heterodimers ((ScFv-Fc/Fc).
- lane M is the molecular weight standard (the top 3
- the fragments were 104KD, 78KD, 50KD from top to bottom, and lanes 1-9 were the mutation combinations 0-8 in Table 5, respectively.
- Figure 6 is a SDS-PAGE diagram of the 31-day accelerated stability test. Wherein Control is wild type, scFv-Fc/Fc-mixl is mutation combination 1, scFv-Fc/Fc-mixl3 is mutation combination 4, and scFv-Fcmixl/Fc-mix2 is mutation combination 6.
- Figure 7 is a CE-SDS diagram of the 31 day accelerated stability test. Control is wild type, Mixl is mutation combination 1, and Mix2 is mutation combination 6. detailed description
- a total of 48 human IgGl antibody crystal structures containing the Fc region were obtained from the protein database (PDB, www.pdb.org) by a structural similarity search algorithm (Reference: Yuzhen Ye and Adam Godzik. FATCAT: a web server for flexible structure Nucleic Acids Res., 2004, 32 (Web Server issue): W582-585. ), the Fc fragment of these 48 antibodies was derived from 1DN2 (PDB numbering).
- the protein contact amino acid recognition software CMA http://ligin.weizmann.ac.il/cma/ was used to screen and identify the antibody (PDB number: 1DN2) in CH3-CH3 according to the distance of the basal acid action.
- the interface aryl acid refers to some of the amino acids whose distance between the heavy chain of the side chain and the heavy atom of any of the other basic acids of the other chain is less than a threshold.
- the threshold value of this embodiment is 4.5A or 5.5 (for example, B. Erman, I. Bahar and RL Jernigan. Equilibrium states of rigid bodies with multiple interaction Sites. Application to protein helices. J. Chem. Phys.
- Table 1 shows 34 interface acids of antibody 1DN2 screened by contact with an aryl acid (i.e., at a distance of less than 4.5 A), wherein chain A and chain B represent the first and second strands of antibody 1DN2, respectively.
- the following acid sites are named according to the EU index of the KABAT number of the antibody Fc.
- Table 1. CH3-CH3 interface amino acid list of antibody 1DN2
- the amino acids of both strands of the antibody are Mutations were obtained, such as the paired amino acid Glu356A- Lys439B.
- the two types of mutations are: 1) mutating Glu356A to Lys356A or Arg356A, and/or mutating Lys439A to Glu439A or Asp439A;
- the opposite charge means that the positively charged acid (lysine (Lys, K) or arginine (Arg, R) or the group of acid (His, H) is mutated to negatively charged acid (aspartic acid ( Asp, D) or glutamic acid (Glu, E)) or mutate negatively acidic (aspartic acid or glutamic acid) to a positively charged acid (lysine or arginine).
- the detailed mutation positions are shown in Tables 3 and 4.
- Table 3 Mutant amino acids in the A chain
- the recognition sequence containing Hind ⁇ and EcoR I at both ends and the protective base are synthesized by SEQ ID NO: 1.
- the human Fc gene full length 780 bp, designated SP-Fc
- pcMVp the mammalian cell expression vector
- the vector backbone was ligated to obtain the recombinant vector pcMVp-SP-Fc (the structure is schematically shown in Figure 1), and the recombinant vector pcMVp-Fc was confirmed by sequencing to insert the SEQ ID between the EcoR I and Hind ⁇ sites of the vector pcMVp.
- amino acid sequence of the Fc protein encoded by nucleotide sequences 16 to 771 in SEQ ID NO: 1 is as follows (as shown in SEQ ID NO: 2):
- the ScFv-Fc fusion white amino acid sequence encoded by nucleotide sequences 16 to 1488 of SEQ ID NO: 3 is as follows (as shown in SEQ ID NO: 4):
- the > ⁇ column of the underlined portion of SEQ ID NO: 2 and SEQ ID NO: 4 is the same.
- NA NA D399K/K409D D204K/K214D indicates wild type without mutation
- NA indicates no mutation at the paired cyano acid site
- "/, indicates the relationship between ",” and "356" in "E356K” Indicates the position of the mutant amino acid.
- E before 356 indicates that the acid before the mutation is E
- K after 356 indicates that the acid after the mutation is K. Others are the same.
- the corresponding expression vectors of the 8 mutations in step 3 were transfected into 293H cells (ATCC CRL-1573) in suspension culture with PEI, and the ratio of pcDNA3.1-zeo-ScFv-Fc and pcMVp-SP-Fc plasmid was converted to 1:1, after 3-4 days of culture, collect the supernatant of the cells.
- Detection principle of protein or antibody mixture composition The fusion protein ScFv-Fc has a larger molecular weight than Fc, then homodimer (ScFv-Fc/ScFv-Fc and Fc/Fc) during the mixing of ScFv-Fc and Fc And the heterodimer (ScFv-Fc/Fc) has different band positions in SDS-PAGE, and the ratio of homodimer to heterodimer can be detected by this principle, and the expression vector of ScFv-Fc and Fc Co-transfection, homodimers (ScFv-Fc/ScFv-Fc, Fc/Fc) and heterodimers (ScFv-Fc/Fc) will eventually occur simultaneously. Table 6. Proportion of homodimers and heterodimers on SDS-PAGE for each mutant
- the pcDNA3.1-zeo-ScFv-Fc and pcMVp-SP-Fc plasmids were transfected with PEI to 293H cells in suspension culture at a ratio of 4:1, 1:1 and 1:4, respectively, using mutation combination 1 (ATCC CRL- 1573), after 3-4 days of culture, the cell supernatant was collected.
- the present invention can change the ratio of the homodimer itself to some extent by changing the co-rotation ratio of the different two-stranded plasmids of Fc, but does not significantly change the proportion of the homodimer as a whole, nor can it be significant. Changing the ratio of heterodimers indicates that the ratio of the homodimer as a whole is relatively stable even by changing the ratio of plasmid co-rotation.
- Mutant Combination 1 and Mutant Combination 6 showed very high stability as well as the wild-type (scFV/scFV homodimeric, control) antibody mixture, starting at 16 days, ScFV - Fc/ScFV-Fc homodimer has partial degradation, while Fc/Fc homodimer maintains superior stability to 31 days.
- mutation combination 1 mutation combination
- mutation combination 2 mutation combination
- mutation combination 4 the antibody produced by mutation combination 4 is mixed.
- the compound has relatively obvious degradation and precipitates in the solution. It is speculated that this mutation combination has five mutations in the Fc single strand, and there are 10 mutations in the Fc dimer, which have a great influence on the structure, and thus the stability changes.
- the relevant results are shown in Figure 6.
- CE-SDS Compared with the traditional SDS-PAGE, CE-SDS has a small sample loading rate, can obtain accurate molecular weight standards, can be on-column detection by ultraviolet light, and can realize quantitative analysis, etc., so that the mixture can be more accurately measured. Degradation of the body. It can be seen from Fig. 7 and Table 6 that Mutant Combination 1 and Mutant Combination 6 also showed sufficient stability with the wild type (control) antibody mixture, and the ScFV-Fc/ScFV-Fc was identical to the first one at 16 days. The polymer degradation peak, the correlation results are consistent with the SDS-PAGE accelerated stability results. Table 6. Accelerated Stability of Antibody Mixtures CE-SDS Results
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EP13822582.6A EP2889313B1 (en) | 2012-07-25 | 2013-07-25 | Method for preparing homodimer protein mixture by using charge repulsion effect |
US14/416,817 US9708389B2 (en) | 2012-07-25 | 2013-07-25 | Method for preparing homodimer protein mixture by using charge repulsion effect |
DK13822582.6T DK2889313T3 (en) | 2012-07-25 | 2013-07-25 | Method of preparing homodimer protein mixture using charge repellent effect |
ES13822582T ES2710936T3 (es) | 2012-07-25 | 2013-07-25 | Método para preparar una mezcla de proteínas homodiméricas usando efecto de repulsión de carga |
JP2015523402A JP6185584B2 (ja) | 2012-07-25 | 2013-07-25 | 電荷の斥力相互作用を使用することによりホモダイマータンパク質の混合物を調製するための方法 |
EP18196046.9A EP3444280A1 (en) | 2012-07-25 | 2013-07-25 | Method for preparing mixture of homodimer proteins by using repulsive interaction of charges |
HRP20190272TT HRP20190272T1 (hr) | 2012-07-25 | 2019-02-11 | Postupak pripreme mješavine homodimera proteina koristeći učinak odbijanja naboja |
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US11130808B2 (en) | 2016-05-26 | 2021-09-28 | Qilu Puget Sound Biotherapeutics Corporation | Mixtures of antibodies |
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CN102851338A (zh) * | 2012-07-25 | 2013-01-02 | 苏州康宁杰瑞生物科技有限公司 | 利用电荷排斥作用制备同二聚体蛋白混合物的方法 |
CN110658340B (zh) * | 2015-01-08 | 2023-10-31 | 苏州康宁杰瑞生物科技有限公司 | 具有共同轻链的双特异性抗体或抗体混合物 |
CN106883297B (zh) | 2015-12-16 | 2019-12-13 | 苏州康宁杰瑞生物科技有限公司 | 基于ch3结构域的异二聚体分子、其制备方法及用途 |
CN110312523B (zh) | 2016-11-08 | 2024-04-26 | 齐鲁皮吉特湾生物治疗有限公司 | 抗pd1和抗ctla4抗体 |
WO2020192648A1 (en) * | 2019-03-25 | 2020-10-01 | Dingfu Biotarget Co., Ltd. | Proteinaceous heterodimer and use thereof |
WO2021000886A1 (zh) | 2019-07-01 | 2021-01-07 | 苏州康宁杰瑞生物科技有限公司 | 百日咳毒素结合蛋白 |
GB202005879D0 (en) * | 2020-04-22 | 2020-06-03 | Petmedix Ltd | Heterodimeric proteins |
WO2022002233A1 (zh) | 2020-07-03 | 2022-01-06 | 苏州康宁杰瑞生物科技有限公司 | 凝血因子xi(fxi)结合蛋白 |
EP4172194A1 (en) | 2020-07-31 | 2023-05-03 | CureVac SE | Nucleic acid encoded antibody mixtures |
CN112977908B (zh) * | 2021-03-02 | 2022-12-02 | 康宁杰瑞(吉林)生物科技有限公司 | 灌装加塞机及其灌装的方法 |
WO2023280092A1 (zh) | 2021-07-05 | 2023-01-12 | 江苏康宁杰瑞生物制药有限公司 | 抗体药物偶联物及其应用 |
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US11130808B2 (en) | 2016-05-26 | 2021-09-28 | Qilu Puget Sound Biotherapeutics Corporation | Mixtures of antibodies |
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CN102851338A (zh) | 2013-01-02 |
JP2015522295A (ja) | 2015-08-06 |
JP6185584B2 (ja) | 2017-08-23 |
CN103388013B (zh) | 2015-03-11 |
HRP20190272T1 (hr) | 2019-04-05 |
US20150274807A1 (en) | 2015-10-01 |
HUE041454T2 (hu) | 2019-05-28 |
EP3444280A1 (en) | 2019-02-20 |
EP2889313A4 (en) | 2016-05-25 |
ES2710936T3 (es) | 2019-04-29 |
DK2889313T3 (en) | 2019-02-18 |
US9708389B2 (en) | 2017-07-18 |
EP2889313B1 (en) | 2018-12-05 |
EP2889313A1 (en) | 2015-07-01 |
CN103388013A (zh) | 2013-11-13 |
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